The purpose of this proposal is to identify the biochemical alterations which mark the onset of irreversible ischemic brain damage. Severe cerebral ischemia, produced in cats, is followed by a period of recirculation of 10-120 min duration. The brain is frozen in situ and, after thin-sectioning, regional mapping techniques (NADH-fluorescence, ATP-luminescence, and pH-umbelliferone fluorescence) are used to locate regions with irreversible depletion of high energy phosphates. In adjacent freeze-dried sections, these regions of energy failures are sampled by micro-dissection and analyzed using quantitative histochemical methods developed by Lowry and coworkers. Measurements of metabolite levels (ATP, phosphocreatine, lactate, NADH, NAD ion, et al.), of tissue ion content (potassium, sodium, and calcium), of tissue enzyme activities (sodium, potassium-ATPase, 5'-nucleotidase, beta-glucuronidase, 2'-3'-cyclic nucleotide phosphodiesterase, et al.), and of lipid composition will be made in regions irreversible damaged by ischemia. These measurements are then compared to those made in areas which are recovering from the ischemic insult, that is, which are resynthesizing ATP. Regional cerebral blood flow will also be determined using the (C-14)-iodoantipyrine technique. By comparing post-ischemic biochemical alterations in recovering vs. irreversibly damaged tissue at various times during recirculation, it will be possible to identify those changes which are critical in the pathobiochemical evolution of ischemic brain damage.